Droplet-based analysis method

Patent metadata

Field	Value
Publication number	US-10512910-B2
Application number	US-201715707908-A
Country	US
Kind code	B2
Filing date	Sep 18, 2017
Priority date	Sep 23, 2008
Publication date	Dec 24, 2019
Grant date	Dec 24, 2019

Abstract

Droplet-based methods of analysis. In an exemplary method, a device having a port connected to a chamber may be selected. A sample-containing fluid may be placed into the port. A pressure differential may be created that drives the sample-containing fluid from the port to the chamber and separates the sample-containing fluid into droplets. A two-dimensional monolayer of the droplets may be formed in the chamber. At least a portion of the monolayer may be imaged.

First claim

We claim:

1. A method of analysis, the method comprising:
selecting a device having a port connected to a chamber;
placing a sample-containing fluid into the port;
creating a pressure differential that drives the sample-containing fluid from the port to the chamber and separates the sample-containing fluid into droplets;
forming a two-dimensional monolayer of the droplets in the chamber; and
imaging at least a portion of the monolayer.

2. The method of claim 1 , wherein the step of placing includes a step of placing the sample-containing fluid as a continuous phase into a well of the device.

3. The method of claim 1 , wherein the droplets are aqueous droplets that are separated from one another by an immiscible carrier liquid.

4. The method of claim 1 , wherein the step of forming includes a step of collecting the droplets in a chamber having a height corresponding to a diameter of the droplets.

5. The method of claim 1 , wherein the step of creating a pressure differential includes a step of applying positive gas pressure or negative gas pressure to a port of the device.

6. The method of claim 1 , further comprising a step of thermally cycling the monolayer to promote nucleic acid amplification in a subset of droplets of the monolayer.

7. The method of claim 1 , wherein the step of imaging includes a step of detecting fluorescence from the monolayer.

8. The method of claim 1 , wherein the step of imaging creates one or more images of droplets of the monolayer, wherein only a subset of the droplets contain an analyte, and wherein the one or more images indicate whether the analyte is present in individual droplets.

9. The method of claim 8 , further comprising a step of determining a number of droplets containing the analyte using the one or more images.

10. The method of claim 8 , wherein the analyte is a nucleic acid target, further comprising a step of amplifying the nucleic acid target within the monolayer.

11. The method of claim 10 , wherein the step of amplifying includes a step of thermally cycling the monolayer to promote nucleic acid amplification in droplets thereof.

12. The method of claim 10 , wherein the droplets contain an intercalating dye or a probe, wherein the probe includes an oligonucleotide labeled with a fluorophore, and wherein the step of imaging includes a step of detecting fluorescence from the intercalating dye or the fluorophore.

13. The method of claim 1 , wherein the chamber lies in a plane and defines an area of the plane, and wherein the step of forming includes a step of covering a majority of the area with droplets.

14. A method of analysis, the method comprising:
selecting a device having a chamber connected separately to a port and a vent;
placing an aqueous fluid into the port;
applying gas pressure to the device to drive the aqueous fluid from the port to the chamber and form partitions of the aqueous fluid, the partitions being separated from one another by a carrier liquid;
amplifying a nucleic acid target in only of a subset of the partitions while the partitions are arranged in a two-dimensional monolayer in the chamber;
imaging at least a portion of the monolayer to create one or more images; and
determining whether individual partitions of the monolayer contain the nucleic acid target using the one or more images.

15. The method of claim 14 , wherein the chamber has a top wall facing a bottom wall, and wherein the chamber has a height measured between the top and bottom walls corresponding to a diameter of the partitions.

16. The method of claim 14 , wherein the chamber lies in a plane and defines an area of the plane, and wherein the step of amplifying is performed while a majority of the area is covered with partitions.

17. The method of claim 14 , wherein the port includes a well, and wherein the step of placing an aqueous fluid includes a step of placing the aqueous fluid into the well.

18. The method of claim 14 , wherein the step of imaging includes a step of detecting fluorescence from the monolayer.

19. The method of claim 14 , wherein the step of amplifying includes a step of thermally cycling the monolayer.

20. The method of claim 14 , wherein the step of applying gas pressure includes a step of applying positive gas pressure or negative gas pressure to the device.

Assignees

• Bio Rad Laboratories

Inventors

• Colston Jr Billy W
• Hindson Benjamin J
• Ness Kevin D
• Masquelier Donald A

Classifications

• G01N2015/0687

  in solutions, e.g. non volatile residue · CPC title

• G01N15/06

  Investigating concentration of particle suspensions (by weighing G01N5/00; investigating sedimentation of particle suspensions G01N15/04; investigating individual particles G01N15/10) · CPC title

• G01N2035/00465

  Separating and mixing arrangements · CPC title

• G01N2035/00237

  Handling microquantities of analyte, e.g. microvalves, capillary networks · CPC title

• G01N35/08

  using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis · CPC title